Abstract:

The invention relates to novel insulin analogs having a basal time-action
profile, which are characterized by the following features: a) the B
chain end consists of an amidated basic amino acid residue such as lysine
or arginine amide; b) the N-terminal amino acid residue of the insulin A
chain is a lysine or arginine radical; c) the amino acid position A8 is
occupied by a histidine radical; d) the amino acid position A21 is
occupied by a glycine radical; and e) one or more substitutions and/or
additions of negatively charged amino acid residues are carried out in
the positions A5, A15, A18, B-1, B0, B1, B2, B3 and B4.

Claims:

1. An insulin analogue of the formula I ##STR00002## whereinA0 corresponds
to Lys or Arg;A5 corresponds to Asp, Gln or Glu;A15 corresponds to Asp,
Glu or Gln;A18 corresponds to Asp, Glu or Asn;B-1 corresponds to Asp, Glu
or an amino group;B0 corresponds to Asp, Glu or a chemical bond;B1
corresponds to Asp, Glu or Phe;B2 corresponds to Asp, Glu or Val;B3
corresponds to Asp, Glu or Asn;B4 corresponds to Asp, Glu or Gln;B29
corresponds to Lys or a chemical bond;B30 corresponds to Thr or a
chemical bond;B31 corresponds to Arg, Lys or a chemical bond;B32
corresponds to Arg-amide, Lys-amide or an amino group; andwherein two
amino acid residues of the group comprising A5, A15, A18, B-1, B0, B1,
B2, B3 and B4 correspond simultaneously and independently of one another
to Asp or Glu.

17. A process for preparing an insulin analogue as claimed in claim 1.

18. The process as claimed in claim 17, wherein a precursor of the insulin
analogue is prepared recombinantly, the precursor is processed
enzymatically to two-chain insulin, and a coupling with argininamide is
carried out in the presence of an enzyme having trypsin activity, and the
insulin analogue is isolated.

19. A method for the manufacture of a medicament for treating diabetes
mellitus comprising the use of an insulin analogue as claimed in claim 1.

20. The method as claimed in claim 19 for the manufacture of a medicament
for the treatment of diabetes mellitus of type I or type II or for
therapeutically assisting beta cell regeneration.

21. A pharmaceutical composition comprising an insulin analogue as claimed
in claim 1 or a physiologically acceptable salt thereof.

22. A formulation of the insulin analogue as claimed in claim 1, wherein
the formulation is in aqueous form comprising the dissolved insulin
analogue.

23. A formulation of the insulin analogue as claimed in claim 1, wherein
the formulation is in the form of a powder.

24. The formulation as claimed in claim 23, wherein the insulin analogue
as claimed in claim 1 is present in crystalline or amorphous form.

25. A formulation of the insulin analogue as claimed in claim 1, wherein
the formulation is in the form of a suspension.

26. A formulation of the insulin analogue as claimed in claim 1, wherein
the formulation additionally comprises a chemical chaperone.

27. A DNA coding for a precursor of an insulin analogue as claimed in
claim 1.

28. A DNA coding for the A chain of an insulin analogue as claimed in
claim 1.

29. A DNA coding for the B chain of an insulin analogue as claimed in
claim 1.

30. A vector comprising a DNA as claimed in claim 27.

31. A vector comprising a DNA as claimed in claim 28.

32. A vector comprising a DNA as claimed in claim 29.

33. A host organism comprising a DNA as claimed in claim 27 or a vector as
claimed in claim 30.

34. A host organism comprising a DNA as claimed in claim 28 or a vector as
claimed in claim 31.

35. A host organism comprising a DNA as claimed in claim 29 or a vector as
claimed in claim 32.

36. A preproinsulin analogue, wherein the C peptide carries the amino acid
residue arginine at its N terminus and two arginine residues or one
arginine residue and one lysine residue on its C terminus, and in the
latter case the lysine residue forms the actual C terminus.

37. The formulation as claimed in claim 22, which additionally comprises a
glucagon-like peptide-1 (GLP1) or an analogue or derivative thereof, or
exendin-3 or -4 or an analogue or derivative thereof.

38. The formulation as claimed in claim 23, which additionally comprises a
glucagon-like peptide-1 (GLP1) or an analogue or derivative thereof, or
exendin-3 or -4 or an analogue or derivative thereof.

39. The formulation as claimed in claim 24, which additionally comprises a
glucagon-like peptide-1 (GLP1) or an analogue or derivative thereof, or
exendin-3 or -4 or an analogue or derivative thereof.

40. The formulation as claimed in claim 25, which additionally comprises a
glucagon-like peptide-1 (GLP1) or an analogue or derivative thereof, or
exendin-3 or -4 or an analogue or derivative thereof.

41. The formulation as claimed in claim 26, which additionally comprises a
glucagon-like peptide-1 (GLP1) or an analogue or derivative thereof, or
exendin-3 or -4 or an analogue or derivative thereof.

42. The formulation as claimed in claim 37, which additionally comprises
exendin-4.

43. The formulation as claimed in claim 42, wherein an analogue of
exendin-4 is selected from a group
comprisingH-desPro36-exendin-4-Lys6-NH2,H-des(Pro36,3-
7)-exendin-4-Lys4-NH2
andH-des(Pro36,37)-exendin-4-Lys5-NH2,or a
pharmacologically tolerable salt thereof.

48. An aqueous formulation of the insulin analogue as claimed in claim 1,
which comprises no zinc or less than 15 μg/ml of zinc.

49. An aqueous formulation of the insulin analogue as claimed in claim 1,
which comprises no zinc or less than 15 μg/ml to 2 mg/ml of zinc.

50. The formulation as claimed in claim 49, where the zinc content is 200
μg/ml.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of International application No.
PCT/EP2009/000,017, filed Jan. 6, 2009, which is incorporated herein by
reference in its entirety; which claims the benefit of U.S. Provisional
Application No. 61/044,659, filed Apr. 14, 2008 and the benefit of
priority of German Patent Application No. 10 2008 003 568.8, filed Jan.
9, 2008 and the benefit of priority of German Patent Application No. 10
2008 025 008.2, filed May 24, 2008.

[0005]The incidence of diabetes has increased in recent years to an almost
epidemic extent. The disorder may result in a serious shortening of life
expectancy. People with diabetes must frequently supply their body with
insulin from outside. It is sensible to optimize the treatment with
insulin. Different insulins with specific pharmacological properties are
now available. In practice, the different insulins are differentiated
according to their duration of action into short-acting insulins,
fast-acting insulins, long-acting insulins and mixed insulins.
Designations used synonymously for long-acting insulins are slow
insulins, depot insulin or else basal insulin. The active ingredients in
many of these insulin products are so-called insulin analogues which have
been derived from human insulin by substitution, deletion and/or addition
of one or more amino acids. The terms "insulin analogues" and "insulins"
are used synonymously herein.

[0006]The policy of intensified insulin therapy attempts to diminish the
health risk by aiming at a stable control of the blood glucose level by
early administration of basal insulins. One example of a current basal
insulin is the medicament Lantus® (active ingredient: insulin
glargine=Gly (A21), Arg (B31), Arg (B32) human insulin). The general aim
of developing novel, improved basal insulins is to minimize the number of
hypoglycemic events. An ideal basal insulin in this connection is one
acting reliably for at least 24 hours in each patient. The insulin effect
ideally has a delayed onset and a time/action profile which is as shallow
as possible, so that the risk of brief hypoglycemia is distinctly
minimized and administration is even possible without previous intake of
foodstuffs. There is a good supply of basal insulin when the insulin
effect persists at the same level for as long as possible, i.e. the body
is supplied with a constant amount of insulin. The risk of hypoglycemic
events is thus low and a patient- and a day-specific variability is
minimized. The pharmacokinetic profile of an ideal basal insulin should
thus be characterized by a delayed onset of action and by a delayed, i.e.
long-lasting and uniform, action.

[0007]However--despite the therapeutic advantages already achieved--none
of the slow insulins described to date shows the pharmacokinetic
properties of an ideal basal insulin. Desirable insulins have such a
shallow and long-lasting time/action profile that the risk of
hypoglycemic events and of the day-dependent variations in the patient is
further minimized and the duration of action is further delayed, so that
it is no longer necessary in some circumstances to administer insulin
daily. This would make simplified treatment of diabetics possible,
especially of elderly diabetics and those in need of care, who are no
longer able to inject insulin themselves, and would thus also be of great
economic benefit. Such basal insulins would additionally be beneficial in
the early phase of type 2 diabetes. Clinicians report that the injection
phobia present in many people deters them from starting insulin therapy
in good time. As a consequence, the control of blood glucose is poor,
leading to the late sequelae of diabetes. A basal insulin which reduces
the number of insulin doses given by injection might have the effect of
making insulin therapy more acceptable to patients.

[0008]Kohn et al. (Peptides 28 (2007) 935-948) describe how it is possible
to optimize the pharmacodynamics of insulin by preparing insulin
analogues whose isoelectric point (pI) is shifted, by addition of lysine
or arginine at the B chain end or at the N terminus of the A and B chain,
in the direction of the alkaline range compared with the isoelectric
point of human insulin (pI=5.6), so that the solubility under
physiological conditions is reduced and a prolonged time/action profile
results. Compound 18 from Kohn et al. (Arg (A0), Gly (A21), Arg (B31),
Arg (B32) human insulin (experimentally determined pI=7.3; calculated
pI=7.58) is described in this connection as the best compound in the
context of the idea. Kohn et al. therefore regard the main aim in
designing novel insulin analogues as being the addition of positively
charged amino acids to the amino acid sequence of human insulin for the
purpose of increasing the isoelectric point from pI=5.6 into the neutral
range.

[0009]This aim in the design of novel insulin analogues is the opposite of
substitution of neutral amino acids in human insulin by acidic amino
acids and/or addition of acidic amino acids, because such a substitution
and/or additions at least partly abolishes the effect of introducing
positively charged amino acids. However, it has now surprisingly been
found that the described desirable basal time/action profile is obtained
with insulin analogues which are characterized by the features that
[0010]the B chain end consists of an amidated basic amino acid residue
such as lysine or argininamide, and [0011]the N-terminal amino acid
residue of the insulin A chain is a lysine or arginine residue, and
[0012]the A8 amino acid position is occupied by a histidine residue, and
[0013]the A21 amino acid position is occupied by a glycine residue, i.e.
in the amidated basic amino acid residue at the B chain end the carboxyl
group of the terminal amino acid is present in its amidated form, and
[0014]there have been two substitutions of neutral amino acids by acidic
amino acids, two additions of negatively charged amino acid residues or
one such substitution and one such addition respectively in the A5, A15,
A18, B-1, B0, B1, B2, B3 and B4 positions.

[0015]Whereas the first three features mentioned tend, through
introduction of positive charges or elimination of negative charges, to
contribute to increasing the pI of a corresponding insulin analogue, the
last-mentioned substitutions and/or additions of negatively charged amino
acid residues have the opposite effect and contribute to reducing the pI.
Surprisingly, precisely the insulin analogues described have the desired
advantageous time/action profiles. The pI values of these compounds are
lower than that of compound 18 from Kohn et al. (Arg (A0), Gly (A21), Arg
(B31), Arg (B32) human insulin), but nevertheless moreover show a delayed
onset of action and a longer duration of action, i.e. an extremely
shallow and long-lasting, uniform action profile. The risk of
hypoglycemic events is thus distinctly minimized. The delay is so marked
that it is surprisingly possible to detect the effect even in model
experiments on rats, although the delayed action of insulin glargine
cannot by contrast be unambiguously observed in rats. FIG. 1 shows the
hypoglycemic effect of the compound YKL205 of the invention compared with
that of insulin glargine. Similar results are obtained in dogs (see FIG.
2). Thus, novel basal insulins which need to be administered distinctly
less frequently have been provided. Besides these pharmacokinetic
advantages described, the analogues of the invention show distinctly
better properties compared with insulin glargine in pharmacological
respects such as, for example, receptor specificity and in vitro
mitogenicity. The claimed insulins also show advantages in
physicochemical respects.

SUMMARY OF THE INVENTION

[0016]The invention thus relates to an insulin analogue of the formula I

##STR00001##

where [0017]A0 corresponds to Lys or Arg; [0018]A5 corresponds to Asp, Gln
or Glu; [0019]A15 corresponds to Asp, Glu or Gln; [0020]A18 corresponds
to Asp, Glu or Asn; [0021]B-1 corresponds to Asp, Glu or an amino group;
[0022]B0 corresponds to Asp, Glu or a chemical bond; [0023]B1 corresponds
to Asp, Glu or Phe; [0024]B2 corresponds to Asp, Glu or Val; [0025]B3
corresponds to Asp, Glu or Asn; [0026]B4 corresponds to Asp, Glu or Gln;
[0027]B29 corresponds to Lys or a chemical bond; [0028]B30 corresponds to
Thr or a chemical bond; [0029]B31 corresponds to Arg, Lys or a chemical
bond; [0030]B32 corresponds to Arg-amide, Lys-amide or an amino
group,where two amino acid residues of the group comprising A5, A15, A18,
B-1, B0, B1, B2, B3 and B4 correspond simultaneously and independently of
one another to Asp or Glu.

[0035]The invention relates in particular to insulin analogues as detailed
above in which independently of one another A0 corresponds to Arg, or
where A5 corresponds to Glu, or where A15 corresponds to Glu, or where
A18 corresponds to Asp, or where B-1 corresponds to an amino group, or
where B0 corresponds to Glu, or where B1 corresponds to Asp, or where B2
corresponds to Val, or where B3 corresponds to Asp, or where B4
corresponds to Glu, or where B29 corresponds to Lys, or where B30
corresponds to Thr, or where B31 corresponds to Arg or Lys.

[0036]The invention particularly preferably relates to an insulin analogue
selected from the group comprising:

[0037]Specification of the term "human insulin" in the designations of the
insulin analogues mentioned makes reference to the amino acid sequences
of the A chain and B chain of human insulin, and all deviations
(additions, substitutions, deletions) therefrom are indicated in a given
designation of an insulin analogue.

[0038]The invention further relates to a process for preparing an insulin
analogue as mentioned above, in particular where a precursor of the
insulin analogue is prepared recombinantly, the precursor is processed
enzymatically to two-chain insulin, and a coupling with argininamide is
carried out in the presence of an enzyme having trypsin activity, and the
insulin analogue is isolated.

[0039]The invention further relates to a use of an insulin analogue as
described above for the manufacture of a medicament for the treatment of
diabetes, in particular of diabetes of type I or type II. The invention
likewise relates to a use of an insulin analogue as described above for
the manufacture of a medicament for assisting beta cell regeneration.

[0040]The invention further relates to a pharmaceutical comprising an
insulin analogue as described above and/or physiologically acceptable
salts thereof.

[0041]The invention further relates to a formulation of the insulin
analogue as described above, where the formulation is in aqueous form
comprising the dissolved insulin analogue.

[0042]The invention further relates to a formulation of the insulin
analogue as described above, where the formulation is in the form of
powder.

[0043]The invention further relates to a formulation as described above,
where the insulin analogue as described above is present in crystalline
and/or amorphous form.

[0044]The invention further relates to a formulation of the insulin
analogue as described above, where the formulation is in the form of a
suspension.

[0045]The invention further relates to a formulation of the insulin
analogue as described above, where the formulation additionally comprises
a chemical chaperone.

[0046]The invention further relates to a DNA coding for a precursor of an
insulin analogue as described above, or for the A chain or B chain of an
insulin analogue as described above.

[0047]The invention further relates to a vector comprising a DNA as
described above.

[0048]The invention further relates to a host organism comprising a DNA as
described above or a vector as described above.

[0049]The invention further relates to a preproinsulin analogue, wherein
the C peptide carries the amino acid residue arginine at its N terminus
and two arginine residues or one arginine residue and one lysine residue
on its C terminus, and in the latter case the lysine residue forms the
actual C terminus.

[0050]The invention further relates to a formulation as described above
which additionally comprises also a glucagon-like peptide-1 (GLP1) or an
analogue or derivative thereof, or exendin-3 or -4 or an analogue or
derivative thereof, preferably exendin-4.

[0051]The invention further relates to a formulation as described above in
which an analogue of exendin-4 is selected from a group comprising

[0055]The invention further relates to a formulation as described above
which additionally comprises Arg34, Lys26
(N.sup.ε(γ-glutamyl(N.sup.α-hexadecanoyl))) GLP-1
(7-37) [liraglutide] or a pharmacologically tolerable salt thereof.

[0056]It is clear to a skilled worker in this connection that the insulins
of the invention may be item of a pharmaceutical formulation which has an
advantageous effect after administration. Aqueous solutions are the
starting point in this connection. Further components must accordingly be
miscible. The risk of viral animal contamination is minimized in that the
preparation ought not to comprise any components derived from animal
sources. It is further advantageous to prevent microbial contamination by
adding preservatives. It is possible by adding isotonic agents to
compensate for a possible negative effect of the formulation on the
physiology of the tissue cells at the administration site. The addition
of protamine may have a stabilizing effect, so that substantially
salt-free insulin preparation can be obtained by adding protamine to the
formulation. Addition of a phenolic component may lead to stabilization
of the structure of the insulin analogue used and thus additionally bring
about inter alia the delaying effect on the onset of action. It is also
possible to add to the formulation substances which stabilize the spatial
structure of the slow insulins of the invention and lead to better
thermal stability. Such chemical chaperones may be for example short
synthetic peptides, which may also comprise amino acid analogues or
include for example peptide sequences derived from the C peptide of
insulin.

[0057]The insulins of the invention can be incorporated into nanoparticles
for developing depot forms. Also conceivable are so-called slow release
formulations in which the slow insulin of the invention is present
reversibly bound to a polymer carrier.

[0058]The insulins of the invention can be administered in parallel with
fast-acting insulin such as insulin glulisine (APIDRA®),
NovoRapid®, insulin lispro (HUMALOG®) or insulin derivatives
undergoing development or formulations with an appropriate time/action
profile or inhalable insulin or nasally or orally administered insulins
which are undergoing development. It will be clear to a skilled worker in
this connection that appropriately formulated mixtures of fast-acting and
slow insulin of the invention can also be used for this purpose. The
insulin analogues of the invention can further be used in pharmaceutical
preparations which comprise peptides which are described by an activity
comparable to GLP-1 (glucagon like Peptide-1) or exendin-4 or exendin-3.
GLP-1 (7-37), exenatide (BYETTA®) or peptides whose preparation is
described in the patent applications WO 2006/058620, WO 2001/04156, WO
2004/005342 and WO 98/08871 represent examples of such peptides.
Formulations particularly advantageous in this connection are those
comprising a depot formulation of these peptides. Types of therapy
advantageous especially in the initial phase of type II diabetes are
those which provide in parallel with the administration of the
pharmaceuticals of the invention, which increase the effect of insulin,
such as, for example, metformin. Combination therapies with dipeptidyl
peptidase-4 inhibitors which increase the level of incretins are, like
combinations with sulfonylureas which increase insulin secretion in the
pancreas, likewise possible. The slow insulins of the invention can be
employed particularly advantageously when regeneration of pancreatic beta
cells from appropriate stem cells is initiated by administration of
differentiation factors. All these applications are mentioned by way of
example for the therapy of diabetes, and the invention likewise relates
thereto. The invention thus further relates to the use of the insulins of
the invention in combination with other active ingredients for the
treatment of diabetes, especially diabetes of type I or type II diabetes.

[0059]The invention further relates to a pharmaceutical which comprises an
insulin analogue of the invention which represents in particular an
aqueous formulation or a powder.

[0060]The pharmaceutical is a pharmaceutical preparation which is
preferably a solution or suspension for injection purposes; it is
characterized by a content of at least one insulin analogue of the
invention, and/or at least one of the physiologically tolerated salts
thereof in dissolved, amorphous and/or crystalline--preferably in
dissolved--form.

[0061]The preparation preferably has a pH of between about 2.5 and 8.5, in
particular between 4.0 and 8.5, preferably comprises a suitable tonicity
agent, a suitable preservative and, where appropriate, a suitable buffer,
and preferably also a particular zinc ion concentration, in sterile
aqueous solution. The totality of the preparation ingredients apart from
the active ingredient forms the preparation carrier. Suitable tonicity
agents are for example glycerol, glucose, mannitol, NaCl, calcium or
magnesium compounds such as CaCl2 etc. The solubility of the
insulins of the invention or the physiologically tolerated salts thereof
at weakly acidic pH values is influenced by the choice of the tonicity
agent and/or preservative.

[0063]Buffer substances which can be used in particular for adjusting a pH
between about 4.0 and 8.5 are for example sodium acetate, sodium citrate,
sodium phosphate etc. Otherwise, physiologically acceptable dilute acids
(typically HCl) or alkalis (typically NaOH) are also suitable for
adjusting the pH.

[0064]If the preparation has a zinc content, preference is given to one of
from 1 to 2 mg/ml, in particular from 1 μg/ml to 200 μg zinc/ml.

[0065]The action profile of the insulin analogues of the invention can
surprisingly be influenced satisfactorily by adding Zn. This allows
preparations which differ in relation to the total duration of action,
the speed of onset of action and the profile of the effect curve and thus
allow individual stabilization of the patient. Another possibility arises
through the use of a "two-chamber insulin device" which allows a
formulation with a rapid onset of action and/or slow gradual onset of
action to be administered depending on the life situation.

[0066]For the purpose of varying the active ingredient profile of the
preparation of the invention it is also possible to admix unmodified
insulin, preferably bovine, porcine or human insulin, especially human
insulin, or insulin analogues and derivatives thereof. It is likewise
possible to admix one or more exendin-4 derivatives or peptides which are
characterized by an activity comparable to GLP-1 (glucagon like
peptide-1) or correspond directly to GLP-1. The invention likewise
relates to such pharmaceuticals (preparations).

[0067]Preferred active ingredient concentrations are those corresponding
to about 1-1500, more preferably about 5-1000 and in particular about
40-400 international units/ml.

[0068]The insulin analogues of the invention are initially prepared
biotechnologically as precursor which does not yet include the amide. The
skilled worker is familiar with a large number of possibilities for
preparing insulins. Host cell systems used in this connection are
bacteria, yeasts and plants or plant cells for cultivation by
fermentation. If cost considerations permit, expression systems which use
animal cells as host system are also conceivable. However, the
precondition therefor is reliable freedom from animal viruses. It is thus
clear that the expression systems described by way of example represent
only a small segment of the host/vector systems developed for the
recombinant preparation of proteins. For example, biotechnological
processes based on yeast or plant systems such as mosses, algae or higher
plants such as tobacco, pea, safflower, barley, corn or oilseed rape are
not described in the application. Nevertheless, the invention likewise
includes host/vector systems and coding DNA sequences which allow the
target peptides to be prepared in appropriate biotechnological expression
systems. Host organisms can thus be selected in particular from the plant
kingdom from organisms of the first division Schizophyta comprising
Schizomycetes, bacteria or blue algae, organisms of the 2nd division
Phycophyta class V Chlorophyceae, organisms of the 2nd division
Phycophyta class VII Rhodophyceae, organisms of the 3rd division
Mycophyta, organisms of the 5th division Bryophyta and organisms of
the 7th division Spermatophyta.

[0069]European patent application EP-A 1 222 207 describes a plasmid
pINT358d which codes for a preproinsulin which includes a modified C
peptide. It is now possible with the aid of the polymerase chain reaction
(PCR) to modify the proinsulin-encoding sequence specifically so that it
is possible to express preproinsulins which can serve as precursors of
the insulins of the invention. Corresponding fusion proteins need not
necessarily be prepared intracellularly. It is clear to the skilled
worker that such proteins can also be prepared by bacterial expression
with subsequent secretion into the periplasm and/or into the culture
supernatant. European patent application EP-A 1 364 029 describes this by
way of example. The invention likewise relates to the proinsulin
precursors which lead to the analogues of the invention.

[0070]The proinsulins prepared in this way can in principle be converted
into an insulin analogue precursor which includes lysine or arginine in
position A0 and carries lysine or arginine at the C-terminal end of the B
chain.

[0071]If the proinsulins of the invention are in the form of inclusion
bodies or soluble form after intracellular expression in bacteria, these
precursors must be folded by in vitro folding into the correct
conformation before the processing and biochemical modification can be
undertaken. In this connection, the described fusion protein allows
direct folding after denaturation by means of urea or guanidinium
hydrochloride, and the invention likewise relates to folding
intermediates.

[0072]Biochemical methods are used to concentrate the individual
intermediates, especially separation processes whose underlying
principles are published and in fact the subject of textbooks. It is
clear to the skilled worker that such principles can consequently be
combined and thus may lead to processes which have not previously been
published in their sequence. The invention thus likewise relates to
processes which lead to purification of the analogues of the invention.

[0073]The invention further relates to a process for preparing the insulin
analogues of the invention, where a precursor of the insulin analogue is
prepared recombinantly and converted enzymatically into a two-chain
insulin precursor which carries arginine or lysine N-terminally in
relation to amino acid 1 of the A chain, and has at the C-terminal end of
the B chain a lysine or arginine residue which is converted with
argininamide or lysinamide in the presence of an enzyme having trypsin
activity into the amide and thus into the slow insulin of the invention,
and is prepared with high purity by a biochemical purification process.

[0074]Proteins which differ through substitution of at least one naturally
occurring amino acid residue by other amino acid residues and/or addition
and/or deletion of at least one amino acid residue from the
corresponding, otherwise identical naturally occurring protein are
referred to as "analogues" of proteins. It is also possible in this
connection for the added and/or replaced amino acid residues to be ones
which do not occur naturally.

[0075]Proteins which are obtained by chemical modification of certain
amino acid residues of initial proteins are referred to as "derivatives"
of proteins. The chemical modification may consist for example of
addition of one or more particular chemical groups to one or more amino
acids.

[0079]FIG. 4: Zinc dependence of the hypoglycemic effect of YKL205 in dogs

[0080]The following examples are intended to illustrate the concept of the
invention without having a restrictive effect in this connection.

Example 1

Preparation of the Vector Derivative pINT3580 which Codes for Gly
(A21)-Insulin and a Modified C Peptide which Carries Arg Arg at the C/A
Chain Boundary

[0081]European patent application EP-A 1 222 207 describes the plasmids
pINT358d, pINT91d and the primer sequence Tir. DNA of these products is
used to construct the plasmid pINT3580. The plasmid pINT358d is moreover
characterized by a gene sequence which codes for a modified C peptide
having particular properties. Three primer sequences are synthesized:

[0084]The codon for the arginine to be introduced is in bold print in both
primers. A PCR is carried out in accordance with the European patent
application EP-A 1 222 207 with each of the primer pairs
Tir/arg_cjunc_rev and arg_cjuncf/pint3580_glya21 rev and with DNA of the
plasmid pINT358d as template. Aliquots of the products of the two
reactions are combined and employed together with the primer pair
Tir/pint3580_glya21 rev in a third PCR. The product of this reaction is
purified after fractionation of the reaction mixture by gel
electrophoresis and is digested with the restriction enzymes Sal1/Nco1 in
accordance with the manufacturer's instructions in one and the same
reaction, the reaction mixture is fractionated by gel electrophoresis,
and the DNA fragment encoding the proinsulin sequence is isolated. The
fragment is then inserted by a DNA ligase reaction into the
Nco1/Sal1-opened pINT91d vector DNA.

[0085]The ligation mixture is used to transform competent E. coli
bacterial cells. The transformation mixture is taken out on selection
plates which contain 25 mg/l ampicillin. Plasmid DNA is isolated from
colonies and characterized by DNA sequence analysis. Correct plasmids are
called pINT3580.

Example 2

Construction of the Plasmid pINT3581 Coding for His (A8), Gly
(A21)--preproinsulin

[0086]The construction takes place as described in example 1 by 3
polymerase chain reactions. The product of the third reaction is inserted
after Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA.
The primers Tir and pint3580_glya21 rev are used. Two further primers are
synthesized:

[0087]The codon which codes for histidine in position 8 of the A chain is
emphasized by emboldening in each case. The construction is carried out
as described in example 1. Template for PCR1 and 2 is DNA of the plasmid
pINT3580. PCR1 is carried out with the primer pair Tin/pint3580_Ha8rev
and PCR2 is carried out with the primer pair
pint3580_Ha8f/pint3580_glya21 rev. The primer pair Tir/pint3580_glya21
rev is employed in PCR 3. Template in this case is a mixture of the
reaction products of PCR1 and PCR2. Correct plasmids are called pINT3581.

Example 3

Construction of the Plasmid pINT3582 Coding for His (A8), Glu (A5), Gly
(A21)--preproinsulin

[0088]The construction takes place as described in example 1 and 2 by 3
polymerase chain reactions. The product of the third reaction is inserted
after Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA.
The primers Tir and pint3580_glya21 rev are used. Two further primers are
synthesized.

[0089]The codon which codes for glutamic acid in position 5 of the A chain
is emphasized by emboldening in each case. The construction is carried
out as described in example 1. Template is DNA of the plasmid pINT3581.
Correct plasmids are called pINT3582.

Example 4

Construction of the Plasmid pINT3583 Coding for His (A8), Asp (A18),
Gly(A21)--Preproinsulin

[0090]The construction differs from example 1 by taking place by only one
polymerase chain reaction. The product of this reaction is inserted after
Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA. The
primer Tir is used. One further primer is synthesized:

[0092]The construction differs from example 1 by taking place by only one
polymerase chain reaction. The product of this reaction is inserted after
Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA. The
primer Tir. pint3580_Dal8rev (ex. 4) is used. Template is DNA of the
plasmid pINT3582. Correct plasmids are called pINT3584. The preproinsulin
encoded by the plasmid is precursor for the compound YKL205-1 which
results after amidation with argininamide and describes the following
structure:

Construction of the Plasmid pINT3585 Coding for His (A8), Glu (A15), Gly
(A21)--Preproinsulin

[0094]The construction differs from example 1 by taking place by only one
polymerase chain reaction. The product of this reaction is inserted after
Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA. The
primer Tir is used. One further primer is synthesized:

[0096]The construction differs from example 1 by taking place by only one
polymerase chain reaction. The product of this reaction is inserted after
Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA. The
primer Tir is used. One further primer is synthesized:

[0097]The codon for glutamic acid in position 15 of the A chain and
aspartic acid in position A18 of the A chain is emphasized by emboldening
in each case. Template is DNA of the plasmid pINT3581. Correct plasmids
are called pINT3586. The preproinsulin encoded by the plasmid is
precursor for the compound YKL205 which results after amidation with
argininamide and describes the following structure:

[0099]The construction differs from example 1 by taking place by only one
polymerase chain reaction. The product of this reaction is inserted after
Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA. The
primer Tir and pint3580_Ea15rev shown in example 6 is used. Template is
DNA of the plasmid pINT3582. Correct plasmids are called pINT3587. The
preproinsulin encoded by the plasmid is precursor for the compound
YKL205-2 which results after amidation with argininamide and describes
the following structure:

Construction of the Plasmid pINT3588 Coding for His (A8), Gly (A21), Asp
(B3)--Preproinsulin

[0101]Construction takes place as described in example 1 and 2 by 3
polymerase chain reactions. The product of the third reaction is inserted
after Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA.
The primers Tir and pint3580_glya21 rev are used. Two further primers are
synthesized:

[0102]The codon which codes for aspartic acid in position 3 of the insulin
B chain is emphasized by emboldening in each case. Construction is
carried out as described in example 1. Template is DNA of the plasmid
pINT3581. Correct plasmids are called pINT3588.

[0106]Carrying out the reactions as described in example 9 but using DNA
of the plasmid pINT3585 as template in PCR1 and PCR2 results in plasmid
pINT3590. The preproinsulin encoded by the plasmid is precursor for the
compound YKL205-4 which results after amidation with argininamide and
describes the following structure:

[0108]Carrying out the reactions as described in example 9 but using DNA
of the plasmid pINT3586 as template in PCR1 and PCR2 results in plasmid
pINT3591. The preproinsulin encoded by the plasmid is precursor for the
compound YKL205-5 which results after amidation with argininamide and
describes the following structure:

[0110]Construction takes place as described in example 1 and 2 by 3
polymerase chain reactions. The product of the third reaction is inserted
after Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA.
The primers Tir and pint3580_glya21 rev are used. Two further primers are
synthesized:

[0111]The codon which codes for aspartic acid in position 3 and glutamic
acid in position 4 of the insulin B chain is emphasized by emboldening in
each case. The construction is carried out as described in example 1.
Template is DNA of the plasmid pINT3581.

[0112]Correct plasmids are called pINT3592. The preproinsulin encoded by
the plasmid is precursor for the compound YKL205-6 which results after
amidation with argininamide and describes the following structure:

Construction of the Plasmid pINT3593 Coding for His (A8), Gly (A21), Glu
(B4)--Preproinsulin

[0114]Construction takes place as described in example 1 and 2 by 3
polymerase chain reactions. The product of the third reaction is inserted
after Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d vector DNA.
The primers Tir and pint3580_glya21 rev are used. Two further primers are
synthesized:

[0115]The codon which codes for glutamic acid in position 4 of the insulin
B chain is emphasized by emboldening. The construction is carried out as
described in example 1. Template is DNA of the plasmid pINT3581. Correct
plasmids are called pINT3593.

[0119]Carrying out the reactions as described in example 9 but using DNA
of the plasmid pINT3585 as template in PCR1 and PCR2 results in plasmid
pINT3595. The preproinsulin encoded by the plasmid is precursor for the
compound YKL205-8 which results after amidation with argininamide and
describes the following structure:

[0121]Carrying out the reactions as described in example 9 but using DNA
of the plasmid pINT3586 as template in PCR1 and PCR2 results in plasmid
pINT3596. The preproinsulin encoded by the plasmid is precursor for the
compound YKL205-9 which results after amidation with argininamide and
describes the following structure:

[0124]There is partial overlap of the two primers in this case.
Pint3581_Eb0f2 contains an NcoI recognition sequence. This is depicted
underlined. The codon which codes for glutamic acid in position 0 at the
start of the B chain is emphasized by emboldening in each case. Template
for PCR1 is DNA of the plasmid pINT3581.

[0125]PCR1 is carried out with the primer pair
pint3581_Eb-1f2/pint3580_glya21 rev. Template for PCR2 is the product
from PCR1. PCR2 is carried out with the primer pair
pint3581_Eb-1f2/pint3580_glya21 rev. The product from PCR2 covers the
complete preproinsulin sequence. The product of the second reaction is
inserted after Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d
vector DNA. Correct plasmids are called pINT3597. Replacement of the
codon for glutamic acid in position B0 by the codon of aspartic acid and
following the example results in plasmids which have aspartic acid
instead of glutamic acid in position B0.

[0126]Carrying out the reactions as described in example 18 but using DNA
of the plasmid pINT3582 as template in PCR1 results in plasmid pINT3598.
The preproinsulin encoded by the plasmid is precursor for the compound
YKL205-10 which results after amidation with argininamide and describes
the following structure:

[0128]Carrying out the reactions as described in example 18 but using DNA
of the plasmid pINT3585 as template in PCR1 results in plasmid pINT3599.
The preproinsulin encoded by the plasmid is precursor for the compound
YKL205-11 which results after amidation with argininamide and describes
the following structure:

[0130]Carrying out the reactions as described in example 18 but using DNA
of the plasmid pINT3586 as template in PCR1 results in plasmid pINT3600.
The preproinsulin encoded by the plasmid is precursor for the compound
YKL205-12 which results after amidation with argininamide and describes
the following structure:

[0133]There is partial overlap of the two primers in this case.
Pint3581_Db-1f2 contains an NcoI recognition sequence. This is depicted
underlined. The codon which codes for aspartic acid in position 1 of the
B chain is emphasized by emboldening in each case. Template for PCR1 is
DNA of the plasmid pINT3581. PCR1 is carried out with the primer
pint3581_Dblfl/pint3580_glya21 rev. Template for PCR2 is the product from
PCR1. PCR2 is carried out with the primer pair
pint3581_Dblf2/pint3580_glya21 rev. The product from PCR2 covers the
complete preproinsulin sequence. The product of the second reaction is
inserted after Nco1/Sal1 cleavage into the Nco1/Sa11-opened pINT91d
vector DNA. Correct plasmids are called pINT3601.

[0134]Carrying out the reactions as described in example 22 by using DNA
of the plasmid pINT3582 as template in PCR1 results in plasmid pINT3602.
The preproinsulin encoded by the plasmid is precursor for the compound
YKL205-13 which results after amidation with argininamide and describes
the following structure:

[0136]Carrying out the reactions as described in example 22 by using DNA
of the plasmid pINT3585 as template in PCR1 results in plasmid pINT3603.
The preproinsulin encoded by the plasmid is precursor for the compound
YKL205-14 which results after amidation with argininamide and describes
the following structure:

[0138]Carrying out the reactions as described in example 22 but using DNA
of the plasmid pINT3586 as template in PCR1 results in plasmid pINT3604.
The preproinsulin encoded by the plasmid is precursor for the compound
YKL205-15 which results after amidation with argininamide and describes
the following structure:

[0141]The codon which codes for glutamic acid in position 0 and which
codes for aspartic acid in each case at the start of the B chain is
emphasized by emboldening in each case. Template for PCR1 is DNA of the
plasmid pINT3597. PCR1 is carried out with the primer pair
pint3597_Dblf/pint3580_glya21 rev. Template for PCR2 is the product from
PCR1. PCR2 is carried out with the primer pair
pint3581_Eblf2/pint3580_glya21 rev. The product from PCR2 covers the
complete preproinsulin sequence. The product of the second reaction is
inserted after Nco1/Sal1 cleavage into the Nco1/Sal1-opened pINT91d
vector DNA. Correct plasmids are called pINT3605. The preproinsulin
encoded by the plasmid is precursor for the compound YKL205-16 which
results after amidation with argininamide and describes the following
structure:

[0144]Template for PCR1 and PCR2 is DNA of the plasmid pINT3586. PCR1 is
carried out with the primer pair desB30f/pint3580_glya21 rev and PCR2 is
carried out with the primer pair Tir/desB30rev template. The template
used for PCR3 is an equimolar mixture of the products from PCR1 and PCR2.
The reaction is carried out with the primer pair Tir/pint3580_glya21 rev.
The product from PCR3 covers the complete preproinsulin sequence. The
product of the third reaction is inserted after Nco1/Sal1 cleavage into
the Nco1/Sal1-opened pINT91d vector DNA. The preproinsulin encoded by the
plasmid is precursor for the compound YKL205-17 which results after
amidation with argininamide and describes the following structure:

[0146]The expression is carried out in accordance with example 1 of
European patent application EP-A 1 222 207.

Example 29

Folding of the Proinsulin Derivatives

[0147]The folding takes place in principle by the method described in EP-A
0 668 282

Example 30

Enzymatic Processing of the Folded Preproinsulin to Give the 2-chain
Arg(A0)--Insulin Precursor whose C-terminal B Chain End is Characterized
by Lysine or Arginine

[0148]The enzymatic processing of the folded preproinsulin precursor takes
place as described for example in example 4 of WO91/03550. It proves to
be particularly advantageous in this case to employ the trypsin variant
described in WO 2007/031187 A1.

[0149]Irrespective of the positioning of the additional acidic amino
acids, a standard reaction is carried out as follows: 100 mg of Arg (A0),
Gly (A21), Arg (B31)--insulin analogue are dissolved in 0.95 ml of
argininamide solution (446 g/L), and 0.13 mL of M Na acetate buffer (pH
5.8) and 2 ml of DMF are added. The reaction mixture is cooled to
12° C. and started by adding 0.094 ml of trypsin (0.075 mg, Roche
Diagnostics). The reaction is stopped after 8 h by adding TFA to pH 2.5
and analyzed by HPLC. There is formation of >60% Arg (A0), Gly (A21),
Arg (B31), Arg (B32)--NH2-- human insulin. Addition of trypsin
inhibitor solution is followed by purification of the amidated analogue
in analogy to U.S. Pat. No. 5,656,722.

[0151]In order to test the insulin derivatives of the invention for their
biopharmacological and physicochemical properties, a solution of the
compounds was prepared as follows: the insulin derivative of the
invention was dissolved with a target concentration of 240±5 μM in
1 mM hydrochloric acid with 80 μg/mL zinc (as zinc chloride).

[0153]For this purpose, initially an amount of the freeze-dried material
which is about 30% higher than required on the basis of the molecular
weight and the desired concentration was weighed out. The concentration
present was then determined by analytical HPLC and the solution was
subsequently made up to the volume necessary to achieve the target
concentration with 5 mM hydrochloric acid with 80 μg/mL zinc. If
necessary, the pH was readjusted to 3.5±0.1. After the final analysis
by HPLC to verify the target concentration of 240±5 μM, the
finished solution was transferred by means of a syringe with a 0.2 μm
filter attachment into a sterile vial which was closed with a septum and
a crimped cap. No optimization of the formulations, e.g. in relation to
addition of isotonic agents, preservatives or buffer substances, was
carried out for the short-term single testing of the insulin derivatives
of the invention.

Example 33

Evaluation of the Blood Glucose-lowering Effect of Novel Insulin Analogues
in Rats

[0154]The blood glucose-lowering effect of selected novel insulin
analogues is tested in healthy male normoglycemic Wistar rats. Male rats
receive subcutaneous injection of a dose of 9 nmol/kg of an insulin
analogue. Blood samples are taken from the animals immediately before the
injection of the insulin analogue and at regular intervals up to eight
hours after the injection, and the blood glucose content therein is
determined. The experiment shows clearly (cf. FIG. 1) that the employed
insulin analogue of the invention leads to a distinctly delayed onset of
action and a longer, uniform duration of action.

Example 34

Evaluation of the Blood Glucose-lowering Effect of Novel Insulin Analogues
in Dogs

[0155]The blood glucose-lowering effect of selected novel insulin
analogues is tested in healthy male normoglycemic beagle dogs. Male
animals receive subcutaneous injection of a dose of 6 nmol/kg of an
insulin analogue. Blood samples are taken from the animals immediately
before the injection of the insulin analogue and at regular intervals up
to 48 hours after the injection, and the blood glucose content therein is
determined. The experiment shows clearly (cf. FIG. 2) that the employed
insulin analogue of the invention leads to a distinctly delayed onset of
action and a longer, uniform duration of action.

Example 35

Evaluation of the Blood Glucose-lowering Effect in Dogs with a Dose
Increased Two-fold

[0156]The blood glucose-lowering effect of selected novel insulin
analogues is tested in healthy male normoglycemic beagle dogs. Male
animals receive subcutaneous injection of a dose of 6 nmol/kg and 12
nmol/kg of an insulin analogue. Blood samples are taken from the animals
immediately before the injection of the insulin analogue and at regular
intervals up to 48 hours after the injection, and the blood glucose
content therein is determined. The experiment shows clearly (cf. FIG. 3)
that the employed insulin analogue of the invention has a dose-dependent
effect but that, despite the dose being increased two-fold, the action
profile has a shallow profile, i.e. no pronounced low point (nadir) is
observed. It can be deduced from this that the insulins of the invention
lead to distinctly fewer hypoglycemic events by comparison with known
slow insulins.

Example 36

Evaluation of the Blood Glucose-lowering Effect in Dogs with Different
Zinc Concentrations in the Formulation

[0157]The experiments were carried out as described in Example 35. FIG. 4
shows the result. According to this, the time-effect curve of the insulin
analogue of the invention can be influenced through the content of zinc
ions in the formulation with the same insulin concentration in such a way
that a rapid onset of action is observed with a zero or low zinc content,
and the effect is maintained for 24 hours, whereas a gradual onset of
action is observed with a higher zinc content, and the insulin effect is
maintained for distinctly longer than 24 hours.